These studies examine myelin assembly employing designs directed at intracellular processing of two integral proteins, proteolipid protein (PLP) and myelin associated glycoprotein (MAG). We will use an in vitro system of tissue slices prepared from actively myelinating murine brainstem. Different labeling protocols and specific inhibitors will be used to delineate the intracellular itinerary and the sites of co- and posttranslational modifications, specifically acylation of PLP and glycosylation of MAG. The kinetics of passage through different subcellular organelles (half-lifes and turn-over rates) will reveal whether PLP and MAG follow the same or different intracellular pathways, and whether the same or different mechanisms regulate their intracellular transport. We will employ specific inhibitors of protein glycosylation to block processing of MAG at different stages in order to determine the role of the oligosaccharide moiety in the sorting and trafficking of proteins. The dysmyelinating mouse mutant quaking which is characterized by genetic underexpression of 72p MAG and overexpression of 67p MAG polypeptides, and their abnormal glycosylation will be a useful biological probe to study the regulatory role of MAG in myelinogenesis. The aberrant MAG metabolism may be the primary disorder in quaking. We will study co- and posttranslational processing of MAG polypeptides in cellular organelles to gain insight into their metabolic relationship(s) during assembly. In addition, we will explore the possibility that the primary structure of MAG polypeptides are altered in quaking. We believe that these studies will lead to new concepts regarding the biochemical events controlling myelination. Once the mechanisms of myelin sheath formation are understood, therapies can be developed to potentiate remyelination in such diseases as multiple sclerosis.